Novel VEGF-like factor

ABSTRACT

A novel human gene having a significant homology with a VEGF-C gene, a member of the VEGF family, has been isolated by the PCR method using primers designed based on the sequence of EST that is assumed to be homologous with the C-terminal region of the VEGF-C gene. Mouse and rat genes have been isolated based on the human gene isolated as above. A protein encoded by the above human gene has been isolated by introducing the gene into  Escherichia coli  and expressing it. The isolated protein and genes can be applied to, for example, gene therapy for the VEGF-D deficiency, wound healing, and promotion of collateral vessel formation. Furthermore, VEGF-D protein inhibitors can be used as a novel anticancer drug, etc.

TECHNICAL FIELD

The present invention relates to a protein factor involved inangiogenesis in humans and falls in the field of genetic engineering.

BACKGROUND ART

The process of angiogenesis, in which endothelial cells existing in theinner wall of blood vessels of animals generate new blood vessels, istriggered by transduction of a specific signal. A variety of substancesare reportedly involved in this signal transduction. The most notablesubstance among them is the vascular endothelial growth factor (VEGF).VEGF is a protein factor which was isolated and purified, and canincrease the proliferation of endothelial cells and the permeability ofblood vessels (Senger, D. R. et al., Science 219: 983-985 (1983);Ferrara, N. and Henzel, W. J., Biochem. Biophys. Res. Commun. 161:851-858 (1989)). It has been reported that the human VEGF gene containseight exons and produces four subtypes consisting of 121, 165, 189, or206 amino acid residues, depending on the difference in splicing, whichcauses different secretion patterns (Houck, K. A. et al., Mol.Endocrinol. 5:1806-1814 (1991)). It has also been reported that there isa VEGF-specific receptor, flt-1, and that the binding of VEGF to flt-1is important for the signal transduction (Vries, C. D. et al., Science255: 989-991 (1992)).

Placental growth factor (PlGF) and platelet-derived growth factor (PDGF)have thus far been isolated and are factors related to VEGF. Thesefactors are found to promote proliferation activities of vascularendothelial cells (Maglione, D. et al., Proc. Natl. Acad. Sci. USA 88:9267-9271 (1991); Betsholtz, C. et al., Nature 320: 695-699(1986)). Inaddition, VEGF-B (Olofsson, B. et al., Proc. Natl. Acad. Sci. USA 93:2576-2581 (1996)) and VEGF-C (Lee, J. et al., Proc. Natl. Acad. Sci. USA93: 1988-1992 (1996); Joukov, V. et al., EMBO J. 15, 290-299 (1996))have recently been isolated.

These factors appear to constitute a family, and this may containadditional unknown factors.

It has been suggested that VEGF is involved in not only vascularformation at the developmental stage but also in the pathologicalneovascularization associated with diabetes, rheumatoid arthritis,retinopathy, and the growth of solid tumors. Furthermore, in addition toits vascular endothelial cell growth-promoting effects listed above,VEGF's ability to increase vascular permeability was suggested to beinvolved in the edema formation resulting from various causes. Also,these VEGF family factors may act on not only the blood vessels but alsothe blood cells and the lymphatic vessels. They may thus play a role inthe differentiation and proliferation of blood cells and the formationof lymphatic vessels. Consequently, the VEGF family factors arepresently drawing extraordinary attention for developing useful, noveldrugs.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to isolate a novel proteinbelonging to the VEGF family and a gene encoding the protein. Wesearched for genes having homology to VEGF-C, which is a recently clonedVEGF family gene, against Expressed Sequence Tags (EST) and SequenceTagged Sites (STS) in the GenBank database. As a result, we found an ESTthat was assumed to have homology to the C-terminal portion of VEGF-C.We then designed primers based on the sequence, and amplified andisolated the corresponding cDNA using the 5′ RACE method and the 3′ RACEmethod. The nucleotide sequence of the isolated cDNA was determined, andthe deduced amino acid sequence therefrom revealed that the amino acidsequence had significant homology to that of VEGF-C. Based on thehomology, we have assumed that the isolated human clone is a fourthmember of the VEGF family (hereinafter designated as VEGF-D). We havealso succeeded in expressing the protein encoded by the isolated humanVEGF-D gene in E. coli cells, and have also purified and isolated it.Furthermore, we have succeeded in isolating the mouse and rat VEGF-Dgenes using the isolated human VEGF-D gene.

In particular, the present invention relates to a novel proteinbelonging to the VEGF family and a gene encoding the protein. Morespecifically it relates to

-   -   (1) A protein shown by SEQ ID NO. 1 or having the amino acid        sequence derived therefrom in which one or more amino acids are        substituted, deleted, or added;    -   (2) A protein encoded by a DNA that hybridizes with the DNA        shown by SEQ ID NO. 2;    -   (3) A DNA encoding the protein of (1);    -   (4) A DNA hybridizing with the DNA shown by SEQ ID NO. 2;    -   (5) A vector containing the DNA of (3) or (4);    -   (6) A transformant carrying the vector of (5);    -   (7) A method of producing the protein of (1) or (2), which        comprises culturing the transformant of (6);    -   (8) An antibody binding to the protein of (1) or (2);    -   (9) A method of screening a compound binding to the protein        of (1) or (2), which comprises a step of detecting the activity        of the protein of (1) or (2) to bind to a test sample; and    -   (10) A compound binding to the protein of (1) or (2), wherein        said compound has been isolated by the method of (9).

The protein of the present invention (VEGF-D) has significant homologyto VEGF-C and can be considered to be a fourth factor of the VEGFfamily. Since the major function of VEGF is vascular formation at thedevelopmental stage and VEGF is considered to be involved in thepathological neovascularization associated with diabetes, rheumatoidarthritis, retinopathy, and the growth of solid tumors, the protein ofthe present invention is thought to have similar functions.

A person skilled in the art could prepare functionally equivalentproteins through modifying VEGF-D of the present invention by adding,deleting, or substituting one or more of the amino acids of VEGF-D shownby SEQ ID NO. 1 using known methods. Modifications of the protein canalso occur naturally in addition to the artificial modificationsdescribed above. These modified proteins are also included in thepresent invention. Known methods for adding, deleting, or substitutingamino acids include the overlap extension polymerase chain reaction(OE-PCR) method (Gene, 1989, 77 (1): 51).

The DNA encoding VEGF-D of the present invention, shown by SEQ ID NO. 2,is useful for isolating DNAs encoding the proteins having similarfunctions to VEGF-D in other organisms. For example, a person skilled inthe. art could routinely isolate homologs of human VEGF-D of the presentinvention from other organisms by allowing the DNA shown by SEQ ID NO.2, or part thereof, as a probe, to hybridize with the DNA derived fromother organisms. The DNA that hybridizes with the DNA shown by SEQ IDNO. 2 is also included in the present invention. The other organismsinclude mice, rats, and rabbits.

The DNA encoding a protein that is functionally equivalent to VEGF-Dusually has high homology to the DNA shown by SEQ ID NO. 2. The highhomology used herein means at least 70% or higher, more preferably 80%or higher, and still more preferably 90% or higher of sequence homology.

An example of the hybridization conditions for isolating the DNA havinghigh homology will be given below. Prehybridization is performed inExpressHyb Solution at 68° C. for 30 minutes. The probe labeled with aradioisotope is denatured at 95° C. to 100° C. for 2 to 5 minutes andrapidly chilled on ice. The probe is added to a new ExpressHyb Solution.The blot is transferred to the solution containing the probe and allowedto hybridize under a temperature gradient of 68° C. to 55° C. for 2hours. The blot is washed four times, for 10 minute each, with a 2×SSCsolution containing 0.05% SDS at room temperature. The blot is thenwashed with. a 0.1×SSC solution containing 0.1% SDS at 45° C. for 3minutes. The blot is subjected to autoradioqraphy.

An example of the hybridization conditions for isolating the DNA havingvery high homology will be given below. Prehybridization is performed inExpressHyb Solution at 68° C. for 30 minutes. The probe labeled with aradioisotope is denatured at 95° C. to 100° C. for 2 to 5 minutes andrapidly chilled on ice. The probe is added into a new ExpressHybSolution. The blot is transferred into the solution containing theprobe, and allowed to hybridize at 68° C. for 1 hour. The blot waswashed four times, for 10 minute each, with a 2×SSC solution containing0.05% SDS at room temperature. The blot was then washed with a 0.1×SSCsolution containing 0.1% SDS at 50° C. for 40 minutes, during which thesolution was replaced once. The blot was then subjected toautoradiography.

Note that the hybridization condition can vary depending on the lengthof the probe (whether it is an oligomer or a probe with more thanseveral hundred bases), the labeling method (whether the probe isradioisotopically labeled or non-radioisotopically-labeled), and thetype of the target gene to be cloned. A person skilled in the art wouldproperly select the suitable hybridization conditions. In the presentinvention, it is especially desirable that the condition does not allowthe probe to hybridize with the DNA encoding VEGF-C.

The DNA of the present invention is also used to produce VEGF-D of thepresent invention as a recombinant protein. Specifically, therecombinant protein can be produced in large quantity by incorporatingthe DNA encoding VEGF-D (for example, the DNA shown by SEQ ID NO. 2)into a suitable expression vector, introducing the resulting vector intoa host, and culturing the transformant to allow the recombinant proteinto be expressed.

The vector to be used for producing the recombinant protein is notparticularly restricted. However, vectors such as pGEMEX-1 (Promega) orpEF-BOS (Nucleic Acids Res. 1990, 18(17): p.5322) are preferable.Suitable examples of the host into which the vector is introducedinclude E. coli cells, CHO cells, and COS cells.

The VEGF-D protein expressed by the transformant can be purified bysuitably combining purification treatments such as solubilization with ahomogenizer or a sonicator, extraction by various buffers,solubilization or precipitation by acid or alkali, extraction orprecipitation with organic solvents, salting out by ammonium sulfate andother agents, dialysis, ultrafiltration using membrane filters, gelfiltration, ion exchange chromatography, reversed-phase chromatography,counter-current distribution chromatography, high-performance liquidchromatography, isoelectric focusing, gel electrophoresis, or affinitychromatography in which antibodies or receptors are immobilized.

Once the recombinant protein is obtained, antibodies against it can beprepared using known methods. The known methods include preparingpolyclonal antibodies by immunizing rabbits, sheep, or other animalswith the purified protein, and preparing monoclonal antibodies from theantibody-producing cells of immunized mice or rats. These antibodieswill make it possible to quantify VEGF. Although the antibodies thusobtained can be used as they are, it will be more effective to use thehumanized antibodies to reduce the immunogenicity. The methods ofhumanizing the antibodies include the CDR graft method and the method ofdirectly producing a human antibody. In the CDR Graft method, theantibody gene is cloned from the monoclonal antibody-producing cells andits antigenic determinant portion is transplanted into an existing humanantibody. In the method of directly producing a human antibody, a mousewhose immune system has been replaced by the human immune system isimmunized, similar to ordinary monoclonal antibodies. The VEGF-D proteinor its antibody thus obtained can be administered into the body bysubcutaneous injection or a similar method.

A person skilled in the art could screen compounds that bind to theprotein of the present invention by known methods.

For example, such compounds can be obtained by making a cDNA library ona phage vector (such as λgtll and ZAP) from the cells expected toexpress the protein that binds to the protein of the present invention(such as lung, small intestine, and heart cells of mammals), expressingthe cDNAs on LB-agarose, fixing the expressed proteins onto a filter,preparing the purified protein of the present invention as abiotin-labeled or a fusion protein with the GST protein, and reactingthis protein with the above filter. The desired compounds could then bedetected by west western blotting using streptavidin or an anti-GSTantibody (Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E.,Fischer, R., Drepps, A., Ullrich, A., and. Schlessinger, J. (1991)Cloning of P13 kinase-associated p85 utilizing a novel method forexpression/cloning of target proteins for receptor tyrosine kinases,Cell 65: 83-90). Another method comprises the following steps. First,express the protein of the present invention fused with the SRF bindingdomain or the GAL4 binding domain in yeast cells. Second, prepare a cDNAlibrary which expresses cDNAs fused with the transcription activationdomain of VP16 or GAL4 from the cells expected to express a protein thatbinds to the protein of the present invention. Third, introduce the cDNAinto the above yeast cells. Fourth, isolate the library-derived cDNAfrom the positive clones. Finally, introduce the isolated cDNA into E.coli to allow it: to be expressed. (When a protein that binds to theprotein of the present invention is expressed in yeast cells, thereporter gene is activated and the positive clone can be detected.) Thismethod can be performed using the two-hybrid system (MATCHMAKERTwo-Hybrid System, Mammalian MATCHMAKER Two-Hybrid Assay Kit, orMATCHMAKER One-Hybrid System (all by Clontech) orthe HybriZAP Two-HybridVector System (Stratagene) (Dalton, S. and Treisman, R. (1992)Characterization of SAP-1, a protein recruited by serum response factorto the c-fos serum response element, Cell 68: 597-612). Alternatively,the binding proteins can be screened by preparing a cDNA library fromthe cells expected to express a substance, such as a receptor, whichbinds to the protein of the present invention (for example, vascularendothelial cells, bone marrow cells, or lymph duct cells), introducingit into such cells as COS, detecting the binding of the protein of thepresent invention by itself or labeled with a radioisotope or afluorescence, and cloning proteins that bind to the protein of thepresent invention (Yamasaki, K., Taga, T., Hirata, Y., Yawata, H.,Kawanishi, Y., Seed, B., Taniguchi, T., Hirano, T., and Kishimoto, T.(1988) Cloning and expression of human interleukin-6 (BSF-2/IFN beta2)receptor, Science 241: 825-828, Fukunaga, R., Ishizaka-Ikeda, E., Seto,Y., and Nagata, S. (1990) Expression cloning of a receptor for murinegranulocyte colony-stimulating factor, Cell 61: 341-350). Still anothermethod comprises applying the culture supernatant or the cellularextract of the cells expected to express a protein that binds to theprotein of the present invention onto an affinity column to which theprotein of the present invention has been immobilized, and purifying theproteins specifically bound to the column. In addition, a DNA encodingthe protein that binds to the protein of the present invention can beobtained by determining the amino acid sequence of the binding protein,synthesizing oligonucleotides based on the sequence, and screening acDNA library with the oligonucleotides as probes.

Furthermore, compounds that bind to the protein of the present inventioncan be screened by contacting compounds, a natural substance bank, or arandom phage peptide display library with the immobilized protein of thepresent invention and detecting the molecules bound to the protein.These compounds can also be screened by high throughput screeningutilizing combinatorial chemistry technology (Wrighton, N. C., Farrell,F. X., Chang, R., Kashyap, A. K., Barbone, F. P., Mulcahy, L. S.,Johnson, D. L., Barrett, R. W., Jolliffe, L. K., and Dower, W. J., Smallpeptides as potent mimetics of the protein hormone erythropoietin,Science (United States) Jul. 26 1996, 273: 458-464,Verdine, G. L., Thecombinatorial chemistry of nature, Nature (England) Nov. 7 1996, 384:11-13, Hogan, J. C. Jr. Directed combinatorial chemistry, Nature(England) Nov. 7 1996, 384: 17-19).

VEGF-D of the present invention may be used for gene therapy byintroducing the VEGF-D gene into the body of the patient with the VEGF-Ddeficiency, or expressing the gene in the body. An anti-sense DNA of theVEGF-D gene may also be used to inhibit the expression of the geneitself, thereby suppressing the pathological neovascularization.

Among the many available methods to introduce the VEGF-D gene or itsantisense DNA into the body, the retrovirus method, the liposome method,the cationic liposome method, and the adenovirus method are preferable.

In order to express these genes in the body, the genes can beincorporated into a suitable vector and introduced into the body by theretrovirus method, the liposome method, the cationic liposome method, orthe adenovirus method. Although the vectors to be used are notparticularly limited, such vectors as pAdexlcw and pZIPneo arepreferable.

The present invention may also be applied for diagnosing disorderscaused by abnormalities of the VEGF-D gene, for example, by PCR todetect an abnormality of the nucleotide sequence of the VEGF-D gene.

Furthermore, according to the present invention, the VEGF-D protein orits agonists can be used to heal wounds, promote collateral vesselformation, and aid hematopoiesis by the hematopoietic stem cells, bytaking advantage of the angiogenic effect of the VEGF-D protein. Theantibodies against the VEGF-D protein or its antagonists can be used asthe therapeutic agents for pathological neovascularization, lymphaticdysplasia, dyshematopoiesis, or edemas arising from various causes. Theanti-VEGF-D antibodies can be used for diagnosing diseases resultingfrom abnormal production of VEGF-D by quantifying VEGF-D.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relationship among the VEGF-D gene, the EST sequences,and the primers used for cloning.

FIG. 2 compares the amino acid sequences of EST (H24828) and VEGF-C.

FIG. 3 compares the amino acid sequences deduced from the VEGF-D geneand from the known genes of the VEGF family proteins.

FIG. 4 a shows the hydrophobicity plot of VEGF-D. FIG. 4 b shows theprediction of the cleavage site of the VEGF-D signal peptide.

BEST MODE FOR IMPLEMENTING THE INVENTION

The following examples illustrate the present invention in detail, butare not to be construed to limit the scope of the invention.

EXAMPLE 1 Homology Search by TFASTA Method

The sequence CGPNKELDENTCQCVC (SEQ ID NO. 3) was designed based on theconsensus sequence found in the BR3P (Balbiani ring 3 protein) repeat atthe C-terminus of VEGF-C. The entire ESTs and STS sequences in theGenbank database (as of 29 Feb. 1996) were then searched by the TFASTAmethod (Person and Lipman, Proc. Natl. Acad. Sci. USA85:2444-2448(1988)). The searching conditions used are shown below(Table 1). TABLE 1 Sequences 392,210 Symbols 135,585,305 Word Size 2 Gapcreation penalty 12.0 Gap extension penalty 4.0

As a result, an EST (Accession No. H24828) that is considered to codethe consensus sequence was found. The sequence is one of the ESTsregistered by The WashU-Merck EST Project, and nine out of 16 amino acidresidues were identical. Further searching for UniGene by NCBI based onthis sequence revealed that five registered sequences (T64149, H24780,H24633, H24828, and T64277 (as of 1 Mar. 1996)), including the aboveEST, were considered to be derived from the same gene. T64277 andT64149, as well as H24828 and H24780, are the combination of the 5′sequence and the 3′ sequence of the same clones, and the length of theinsert in both of these clones was 0.9 kb (FIG. 1).

Translating the H24828 sequence into a protein sequence in a frame wherehomology is found suggested that this sequence codes 104 C-terminalamino acid residues. Comparing this amino acid sequence with theC-terminus of VEGF-C, 28 out of 104 amino acids (27%) were identical.Moreover, the amino acids that are important for maintaining the proteinstructure, such as cysteine and proline, were well conserved (FIG. 2).Conserved sequences are shown in a black box.

EXAMPLE 2 cDNA Cloning from a Library

Primers for 5′ RACE and 3′ RACE (5′ RACE primer:5′-AGGGATGGGGAACTTGGAACGCTGAAT-3′ (SEQ ID NO. 4), 3′ RACE primer: 5′-GATCTAATCCAGCACCCCAAAAACTGC-3′ (SEQ ID NO.5)) were designed (FIG. 1). Adouble-stranded cDNA was synthesized from human lung-derived polyA⁺ RNAusing reverse transcriptase. PCR was then performed using Marathon-ReadycDNA, Lung (Chlontech), having an adapter cDNA ligated to both ends as atemplate cDNA, and using the above primer and adapter primer (AP-1primer) as primers. The above adapter cDNA contains the regions to whichthe adapter primers AP-1 and AP-2 hybridize. The PCR was performed in amanner such that the system was exposed to treatment at 94° C. for 1min; five cycles of treatment at 94° C. for 30 sec and at 72° C. for 4min; five cycles of treatment at 94° C. for 30 sec and at 70° C. for 4min; then 25 cycles of treatment at 94° C. for 20 sec and at 68° C. for4 min. (TaKaRa Ex Taq (Takara Shuzo) and the attached buffer were usedas Taq polymerase instead of Advantage KlenTaq Polymerase Mix.) As aresult, 1.5 kb fragments were amplified at the 5′ region and 0.9 kbfragments at the 3′ region. These fragments were cloned with thepCR-Direct Cloning System (Clontech), CR-TRAP Cloning System(GenHunter), and PT7Blue-T vector (Novagen). When the 5′-RACE fragmentwas cloned into the pCR-Direct vector, the fragment was amplified againusing 5′-CTGGTTCGGCCCAGAACTTGGAACGCTGAATCA-3′ (SEQ No. 7) and5′-CTCGCTCGCCCACTAATACGACTCACTATAGG-3′ (SEQ ID NO. 8) as primers.

EXAMPLE 3 Nucleotide Sequence Analysis

ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit withAmplitaq DNA Polymerase FS and 377 A DNA Sequencer (ABI) were used forDNA sequencing. The primers used are the primers in the vectors(5′-AATTAACCCTCACTAAAGGG-3′ (SEQ ID NO. 9), 5′-CCAGGGTTTTCCCAGTCACGAC-3′(SEQ ID NO. 10)), AP-2 primer (5′-ACTCACTATAGGGCTCGAGCGGC-3′ (SEQ ID NO.11)), and 10 primers in the sequence shown below (Table 2). TABLE 2 SQ15′-AAGTCTGGAGACCTGCT-3 (SEQ ID NO. 12) SQ2 5′-CAGCAGGTCTCCAGACT-3′ (SEQID NO. 13) SQ3 5′-CGCACCCAAGGAATGGA-3′ (SEQ ID NO. 14) SQ45′-TGACACCTGGCCATTCCA-3′ (SEQ ID NO. 15) SQ5 5′-CATCAGATGGTAGTTCAT-3′(SEQ ID NO. 16) SQ6 5′-ATGCTGAGCGAGAGTCCATA-3′ (SEQ ID NO. 17) SQ75′-CACTAGGTTTGCGGCAACTT-3′ (SEQ ID NO. 18) SQ85′-GCTGTTGGCAAGCACTTACA-3′ (SEQ ID NO. 19) SQ95′-GATCCATCCAGATCCCTGAA-3′ (SEQ ID NO. 20) SQ105′-CAGATCAGGGCTGCTTCTA-3′ (SEQ ID NO. 21)

Determining the nucleotide sequence of the 1.5 kb fragment at the5′-side and the 0.9 kb fragment at the 3′-side revealed that thesequence of the overlapping region was identical, confirming that 5′-and 3′-side cDNAs of the desired gene were obtained. Determining theentire nucleotide sequence of the cDNA revealed that this novel gene hasthe full length of 2 kb and can code a protein consisting of 354 aminoacid residues (SEQ ID NO. 1 and SEQ ID NO. 2). FIG. 1 shows the relationbetween this gene and the EST sequences registered in the Genbankdatabase. Comparing the amino acid sequence with other VEGF familyproteins revealed that the amino acids that are well conserved betweenfamily proteins are also conserved in this novel gene, and thereforethis gene is obviously a new member of the VEGF family (FIG. 3). In FIG.3, HSVEGF indicates human VEGF; HSVEGF-D, HSVEGF-C, and HSVEGF-Bindicate human VEGF homologues (human VEGF-D, human VEGF-C, and humanVEGF-B, respectively); HSPDGF-A indicates human PDGF-A; HSPDGF-Bindicates human PDGF-B; and HSP1GF2 indicates human PlGF2. The conservedsequences are shown in a black box. Since VEGF-D is highly homologous toVEGF-C that was cloned as the Flt4 ligand, it was presumed to be aligand to a Flt-4-like receptor.

Deducing the signal peptide cleavage site (FIG. 4 b) by hydrophobicityplot (FIG. 4 a) and the method of von Heijne (von Heijne, G, NucleicAcids Res. 14, 4683-4690(1986)), N-terminal 21 amino acid residues maybe cleaved as signal peptides, and they may also undergo additionalprocessing like VEGF-C.

EXAMPLE 4 Northern Blot Analysis

A 1 kb fragment, which had been cut out by digestion with EcoRI from the5′-fragment subcloned into pCR-Direct vector, was labeled with[α-³²P]dCTP and used as a probe. Labeling was performed by randompriming using Ready-to Go DNA labeling beads (Pharmacia). Hybridizationwas performed in ExpressHyb Hybridization Solution (Clontech) by theusual method using Multiple Tissue Northern (MTN) Blot-Human, Human II,Human Fetal, and Human Cell lines (Clontech). Significant expression wasobserved in lung, heart, and intestine. Weak expression was observed inskeletal muscle, ovary, colon, and pancreas. The apparent molecularweight of the mRNA was 2.2 kb, and the cloned fragment seemed to bealmost the full length of the gene.

EXAMPLE 5 VEGF-D Protein Expression in E. coli

Two primers, 5′-TCCAGATCTTTTGCGGCAACTTTCTATGACAT-3′ (SEQ ID NO. 22) and5 ′-CAGGTCGACTCAAACAGGCACTAATTCAGGTAC-3′ (SEQ ID NO. 23), weresynthesized to amplify the region corresponding to the 89th to 181stamino acid residues of human VEGF cDNA. The thus-obtained DNA fragmentwas digested with restriction enzymes BglII and SalI, and ligated usingligation kit II (Takara Shuzo Co., Ltd) to plasmid pQE42 (QIAGEN), whichhad been digested with restriction enzymes BamHI and SalI. The resultingplasmid was introduced into E. coli SG19003[pREP4] (QIAGEN), and aplasmid, which was obtained as designed without any mutation, wasselected (pQE42-BS3). Plasmid pQE42-BS3 was introduced into E. coli BL21(Invitorogen) and cultured in 10 ml of L Broth containing 100 mg/lbicucilline (ampicillin sodium for injection, Meiji Seika Kaisha, Ltd.).200 ml of fresh L Broth was then inoculated with the culture. Afterincubation at 37° C. for 1.5 hours, IPTG was added to 3 mM, and theculture was further incubated at 37° C. for 5 hours. After cells wereharvested, a protein was purified with a Ni-NTA column following theprotocol of QIAexpress TypeII kit.

EXAMPLE 6 Expression of DHFR-VEGF-D Fusion Protein in E. coli

The region corresponding to the 89th to 181st amino acid residues ofhuman VEGF cDNA was amplified with the same primers used in Example 5.The thus-obtained DNA fragment was digested with restriction enzymesBglI and SalI. The fragment was then ligated using ligation kit II(Takara Shuzo Co., Ltd.) to the plasmid pQE40 (QIAGEN), which had beendigested with restriction enzymes BamHI and SalI. The resulting plasmidwas introduced into E. coli SG19003[pREP4] (QIAGEN), and a plasmid,which was obtained as designed without any mutation, was selected(pQE40-BS3). Plasmid pQE40-BS3 was introduced into E. coli BL21(Invitrogen) and cultured in 10 ml of L Broth containing 100 mg/lbicucilline (ampicillin sodium for injection, Meiji Seika Kaisha, Ltd.).200 ml of fresh L Broth was then inoculated with the culture. Afterincubation at 37° C. for 1.5 hours, IPTG was added to 3mM, and theculture was further incubated at 37° C. for 5 hours. After cells wereharvested, a DHFR-VEGF-D fusion protein was purified with a Ni-NTAcolumn following the protocol of a QIAexpress TypeII kit.

EXAMPLE 7 Cloning Mouse VEGF-D cDNA

Two Hybond-N+ (Amersham) filters (20 cm×22 cm) on which 1.5×10⁵ pfu ofMouse lung 5′-stretch cDNA library was transferred were prepared.Gradient hybridization from 68° C. to 55° C. was performed for 2 hoursin ExpressHyb Hybridization Solution (Clontech) using as a probe anapproximately 50 ng Pvu II fragment of human VEGF-D, which had beenlabeled with α³²P-dCTP (Amersham) using Ready-To-Go DNA LabelingBeads(-dCTP) (Pharmacia). The filters were washed four times in 2×SSC,0.05% SDS at room temperature for 10 min, then washed in 0.1×SSC, 0.1%SDS at 45° C. for 3 min. The washed filters were exposed overnight at−80° C. using HyperFilm MP (Amersham) and intensifying paper. Positiveclones were subjected to the second screening in the same manner asabove to isolate a single clone. Isolated lambda DNAs were purified fromthe plate lysate using a QIAGEN Lambda MAX I Kit (Qiagen). Insert DNAswere cut out with EcoRI and subcloned into pUC118 EcoRI/BAP (TakaraShuzo Co., Ltd.). Its nucleotide sequence was then determined withABI377 sequencer (Perkin Elmer). The cDNA coding the full length ofmouse VRGF-D was reconstructed with two of the obtained clones thatoverlapped each other. SEQ ID NO. 24 shows the nucleotide sequence ofmouse VEGF-D cDNA and the deduced amino acid sequence therefrom.

EXAMPLE 8 Cloning Rat VEGF-D cDNA

Two Hybond-N+ (Amersham) filters (20 cm×22 cm), on which 1.5×10⁵ pfu ofRat lung 5′-stretch cDNA library had been transferred, were prepared.Gradient hybridization from 68° C. to 55° C. was performed for 2 hoursin ExpressH.Fyb Hybridization Solution (Clontech) using as a probe anapproximately 1 μg fragment containing 1-782 bp of the mouse VEGF-D cDNAwhich had been labeled with α³²P-dCTP (Amersham) using Ready-To-Go DNALabeling Beads(-dCTP) (Pharmacia). The filters were washed four times in2×SSC, 0.05% SDS at room temperature for 10 min, then washed in 0.1×SSC,0.1% SDS at 45° C. for 3 min. The washed filters were exposed overnightat −80° C. using HyperFilm MP (Amersham) and intensifying paper.Positive clones were subjected to the second screening in the samemanner as above to isolate a single clone. The isolated positive clonewas excised into pBluescript using E. coli SOLAR (Stratagene) and helperphage ExAssist (Stratagene.), then the sequence was determined with.ABI377 sequencer (Perkin Elmer). The sequence seemed to be the ratVEGF-D cDNA but did not contain the termination codon.

To obtain the C-terminal cDNA which had not been obtained, PCR wasperformed using Marathon-Ready rat kidney cDNA (Clontech) as a templateand 5′ primerGCTGCGAGTGTGTCTGTAAA (SEQ ID NO. 26) and 3′ primerGGGTAGTGGGCAACAGTGACAGCAA (SEQ ID NO. 27) with 40 cycles of 94° C. for15 sec, 55° C. for 30 sec, and 72 ° C. for 2 min. After thethus-obtained fragment was subcloned into pGEM-T vector (promega), thenucleotide sequence was determined with ABI377 sequencer (Perkin Elmer).The resulting clone contained the C-terminus of rat VEGF-D. Based on theresults of sequencing the clone obtained by plaque hybridization and theclone obtained by PCR, the full length of the rat VEGF-D sequence wasdetermined. SEQ ID NO. 25 . shows the determined nucleotide sequence andthe deduced amino acid sequence therefrom.

Industrial Applicability

In the present invention, a novel protein (VEGF-D) having significanthomology to VEGF-C and its gene have been isolated. VEGF-D appears to beinvolved in the pathological neovascularization associated withdiabetes, rheumatoid arthritis, the growth of solid tumors,differentiation and proliferation of blood cells, formation of lymphaticvessels, and formation of edema resulting from various causes as well asthe normal neovascularization at the developmental stage. The gene ofthe present invention can be used to diagnos disorders caused byabnormalities of the VEGF-D gene and gene therapy for the VEGF-Ddeficiency. The VEGF-D protein, which is obtained by expressing the geneof the present invention, can be used for healing wounds, promotingcollateral vessel formation, and aiding hematopoietic stem cellproliferation. The antibodies or inhibitors against the VEGF-D proteincan be used for treating angiodysplasia and lymphangiodysplasiaassociated with inflammation, edemas arising from various causes,dyshematopoiesis, and, as a novel anticancer agent, for treatingpathological neovascularization. The VEGF-D protein and its antibodiescan be useful for diagnosing diseases resulting from abnormal productionof VEGF-D.

1. canceled.
 2. canceled.
 3. A DNA encoding a protein shown by SEQ IDNO: 1 or having the amino acid sequence derived therefrom in which oneor more amino acids are substituted, deleted, or added.
 4. A DNAhybridizing with the DNA shown by SEQ ID NO:
 2. 5. A vector containingthe DNA of claim 3 or
 4. 6. A transformant carrying the vector of claim5.
 7. A method of producing a protein shown by SEQ ID NO: 1 or havingthe amino acid sequence derived therefrom in which one or more aminoacids are substituted, deleted, or added or a protein encoded by a DNAhybridizing with the DNA shown by SEQ ID NO:2, wherein said methodcomprises culturing the transformant of claim
 6. 8. canceled. 9.canceled.
 10. canceled.